The ability of certain amine-containing macrocyclic compounds, or their acyclic equivalents, to tightly bind metal ions makes these compounds attractive choices as metal ion chelating agents for use in medical diagnosis or therapy. To facilitate diagnosis or therapy, it is desirable to covalently attach, or conjugate, the metal ion-chelating agent complex to biomolecules, such as antibodies. One method of achieving conjugation to a biomolecule is by introducing an active group into the chelating agent and then reacting the active group with a functional group on the biomolecule. For example, the chelating agent may be functionalized with an active group, such as N-hydroxy succinimide (NHS), that in turn, can react with a functional group on the biomolecule.
There is a longstanding need for the efficient preparation of functionalized chelating agents with active groups that can couple the-chelating agent to biomolecules. For example, the efficient functionalization of macrocyclic compounds, such as 1,4,7,10-tetraazocyclododecane-1,4,7,10 tetraacetic acid (DOTA) and 1,4,8,11-tetraazocyclotetradecane 1,4,8,11-tetraacetic acid (TETA), remains problematic. As discussed in U.S. Pat. Nos. 5,428,156 and 5,639,879 to Mease et al., incorporated herein by reference, previous methods for the preparation of functionalized DOTA or TETA are complex, and may yield mixed anhydride products that are highly sensitive to hydrolysis in water, or products with greater than one active ester attached to the chelating agent. The latter can result in the undesirable crosslinking of two or more biomolecules via a reaction between biomolecules and the active esters attached to a single chelating agent.
In an attempt to address these problems, Mease et al., proposed a method to produce DOTA-NHS and TETA-NHS. Mease et al. starts with commercially available 1,4,7,10-tetraazacyclododecane (Cyclen) (or 1,4,8,11-tetraazacyclotetradecane; Cyclam), which is cyanomethylated to produce a tetranitrile. The tetranitrile is then hydrolyzed and acidified to produce DOTA. DOTA has four equivalent free carboxylate groups (i.e., four acetate groups) attached to the Nitrogen atoms of the 1,4,7,10-tetraazacyclododecane ring. To make DOTA-NHS, Mease et al. then prepared a mixture of DOTA, NHS, and a coupling agent, dicyclohexylcarbodiimide (DCC), using dimethylsulfoxide as the solvent. Mease et al., then reacts this product mixture with a biomolecule to make a DOTA-biomolecule conjugation product. There are several problems with this approach, however.
First, the production of mono-substituted DOTA-NHS may be less than desired. This is expected because all four carboxylate groups of DOTA are available for derivatization, and therefore there is no control over the number or location of N-hydroxysuccinimidyl esters attached to DOTA. It is expected, for example, that if equi-molar amounts of DOTA, NHS and DCC were reacted, then a substantial proportion of di-, tri- and tetra-NHS substituted DOTA compound would be present in the product mixture. To reduce the production of such multi-substituted DOTA-NHS compounds, Mease et al. reacted one molar equivalent of DOTA with 0.5 molar equivalents of DCC and NHS. By doing so, Mease et al. found an average of one carboxylic acid on DOTA reacted with NHS. This approach however, must result in low yields of DOTA-NHS. Specifically, if 1 mole of DOTA is reacted with 0.5 moles of NHS, it follows therefore, that the yield of DOTA-NHS could not exceed 50 mole percent. Moreover, this is not an isolated yield of pure DOTA-NHS.
This raises a second problem. Because NHS-substituted DOTA compounds contain a highly reactive Oxygen-Nitrogen bond that links DOTA to NHS, it would be difficult to isolate mono-substituted DOTA-NHS from a mixture of di-, tri- and tetra-NHS substituted DOTA compounds. It is also expected that standard isolation techniques would cause substantial hydrolysis of mono-substituted DOTA-NHS back to the starting compounds. Alternatively, the use of unpurified mixtures of mono-, di-, tri- and tetra-NHS substituted DOTA is expected to resul in poor yields of DOTA bioconjugated to biomolecules in useful fashion.
A third problem associated with the use of multi-NHS substituted DOTA is the potential of crosslinking two or more biomolecules to each other when more than one biomolecule conjugates to a single DOTA molecule. Such cross-linked biomolecule-DOTA conjugates may have poor uptake to a target location, such as a specific organ or cell types, when delivered to a living system.
There is a fourth problem related to the stability of NHS substituted DOTA. The ester group in NHS-substituted DOTA is highly susceptible to hydrolysis in the presence of water. Therefore, even small amounts of water present or absorbed into the dimethylsulfoxide solvent used by Mease et al. will result in hydrolysis of mono-NHS substituted DOTA, thereby reducing the total amount of NHS substituted DOTA available to react with biomolecules.
Accordingly, what is needed is an improved process for the efficient manufacture of functionalized chelating agent that avoids the problems encountered in previous processes.